Structure based design of capsid stabilizing or antiviral agents

ABSTRACT

Agents having the structure, 
     
         U--CH.sub.2 --V--CH.sub.2 --W, 
    
     and agents having the structure, 
     
         U--CH.sub.2 --V--CH.sub.2 --W--X--Y--Z, 
    
     where U, W, and Z are double fused aromatic rings, V is a single six-membered aromatic ring with a hydroxyl group constituent, X is a polar group, and Y is a positively charged group, are disclosed. The double fused aromatic rings can be a five-membered aromatic ring, or a six-membered aromatic ring, fused to a six-membered aromatic ring; the double fused aromatic rings can comprise heteroatoms. The agents can be used as capsid stabilizing or antiviral agents.

GOVERNMENT SUPPORT

This invention was supported by NIH Grant Nos. AI32480, AI20566 andGM39589 and the United States Government has certain rights to thisinvention.

RELATED APPLICATION

This application is a continuation of application Ser. No. 08/207,411filed Mar. 7, 1994, now abandoned, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Poliovirus is a member of the picornaviruses, a large family of smallribonucleic acid-containing viruses responsible for many serious humanand animal diseases (Rueckert, R. R. Virology, 2nd ed. (Field, B. N. etal., eds.) Raven Press, Ltd., New York, p. 508-548 (1982)). Two generaof the picornavirus family are enteroviruses, which include poliovirusand Coxsackievirus, and rhinoviruses. Poliovirus is the etiologic agentof the disease poliomyelitis in humans, and there are three knownserotypes of the virus. The oral poliovaccine, typically given tochildren, is a mixture of the Sabin strain of the three serotypes of thevirus. Mahoney and Leon (the parent strains of Sabin 1 and 3,respectively) are human neurovirulent strains of poliovirus. The oralpoliovirus vaccine is safe and effective, yet has two limitations.First, the vaccine is unstable; current vaccines are inactivated byrelatively brief (less than 24 hours) exposure to temperatures of 37°C., necessitating transport in a frozen state from the site ofmanufacture to the locale where they are administered. Second, thevaccine occasionally reverts to virulence in vaccine recipients; thereverted virulent virus may also be passed to other individuals who comeinto contact with the recipient in whom the vaccine has reverted. Thehuman rhinoviruses consist of at least 100 serotypes and are the primarycausative agents of the common cold. Because of the large number ofserotypes, development of a vaccine is problematic; antiviral agents maytherefore be the best approach to treatment. Other important members ofthe picornavirus family include human hepatitis A virus, Theiler'smurine encephalomyelitis virus, foot-and-mouth disease virus, andmengovirus.

Several crystal structures of poliovirus and rhinovirus capsids havebeen solved by X-ray diffraction. The X-ray structures of poliovirusP1/Mahoney (Hogle, J. M., et al., Science 229:1358 (1985)); poliovirusP3/Sabin (Filman, D. J., et al., EMBO J. 8:1567 (1989)); rhinovirus 14(Rossman, M. G., et al., Nature 317:145 (1985)); rhinovirus 1A (Smith,T. J., et al., Science 233:1286 (1986)); and rhinovirus 16 (Oliveira, M.A., et al., Structure 1(1):51-68 (1993)) are strikingly similar,although poliovirus and the rhinoviruses are currently classified indifferent genuses. From experiment, it is known that there is a bindingsite in the poliovirus structure which usually binds a lipid-likemolecule (Filman, D. J., et al., EMBO J. 8:1567 (1989)). When a drug isbound in this site, the virus is stabilized, and in some cases,infection is prevented (McSharry, J. J., et al., Virology 97:307 (1979);Smith, T. J., et al., Science 233:1286 (1986); reviewed in Zhang, A., etal., Virology, 3:453 (1992)).

The existing drugs against these viruses are only moderately effective.Available drugs are typically effective against a limited subset of therhinovirus serotypes. In addition, the drugs have either failed todemonstrate sufficient prophylactic effect or are converted in the bodyinto inactive metabolites. Furthermore, current drugs have all beenderived from the same parent compound that was found through large-scalerandom screening of known chemicals for activity against the virus, avery expensive and time-consuming process. A need continues foradditional drugs with better efficacy, and with efficacy against severalviruses.

SUMMARY OF THE INVENTION

The current invention pertains to capsid stabilizing or antiviralagents, having a general structure known as a "core" region. The coreregion consists of three functional groups: a fused double aromatic ringgroup (U), which consists of either a five- or a six-membered aromaticring fused to a six-membered aromatic ring; a single six memberedaromatic ring (V) which can optionally have a hydroxyl group substituenton any of the available ring atoms; and a second fused double aromaticring (W), which also consists of either a five- or a six-memberedaromatic ring fused to a six-membered aromatic ring. The first fuseddouble aromatic ring (U), the second fused double aromatic ring (W), orboth of the fused double aromatic rings (U and W), can have heteroatomssubstituted for carbon atoms. These three functional groups areconnected by single linker carbon atoms, such that the core region hasthe general structure:

    U--CH.sub.2 --V--CH.sub.2 --W.

The agent may additionally have a "tail" region connected to the coreregion. The tail region also consists of three functional groups: apolar group (X) with an oxygen atom, a positively charged group (Y), anda fused double aromatic ring (Z), which may be either a five- or asix-membered aromatic ring fused to a six-membered aromatic ring. Thefused double aromatic ring can have heteroatoms substituted for carbonatoms. The positively charged group can be, for example, an ammoniumgroup. These three functional groups are arranged such that the tailregion has the general structure:

    X--Y--Z.

If such a tail region is present, it is connected to the core regionsuch that the overall structure of the agent is:

    U--CH.sub.2 --V--CH.sub.2 --W--X--Y--Z.

Any of the functional groups can have additional small groups attached,such as, for example, alkyl groups (such as methyl groups) halides,hydroxyl groups or amino groups.

The agents were designed through the use of new computationaltechnologies, which allowed the "mapping" of the drug binding site inthe virus of interest, including the localization of functional groupswithin the binding site. The agents were specifically designed so thatthe functional groups have potential energy minima within the bindingsite.

The agents can be used to increase the thermal stability of the virus,and thereby formulate thermostable virus vaccines, making it possible toadminister the vaccine to humans in areas of the world whererefrigeration is prohibitively expensive. Furthermore, the agents whichdecrease the infectivity of the virus can also be used for earlyintervention in cases of poliomyelitis. Alternatively, the agents can beused to terminate excretion of virus in vaccinees after immuneinduction, as well as for prophylaxis in non-vaccinated individuals whomight become infected by a vaccinee in whom the virus has reverted tovirulent form. In addition, since previous studies have shown thatcapsid binding drugs generally have a broad spectrum of activity amongentero/rhinovirus (Andriew, K. et al., J. Virol. 64:1117-1123 (1990)),it is reasonable to expect that agents which bind to one virus, such aspoliovirus, will be effective against related viruses, such asrhinoviruses and Coxsackieviruses. The agents can also be used tostabilize an unstable form of a virus for experimental studies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the general structure of a core region of theagents of the current invention.

FIG. 2 is a depiction of a representative core region.

FIG. 3 is a depiction of a representative core region.

FIG. 4 is a depiction of a representative core region.

FIG. 5 is a depiction of a representative core region.

FIG. 6 is a depiction of a representative core region.

FIG. 7 is a depiction of the general structure of a tail region of theagents of the current invention.

FIG. 8 is a depiction of a representative tail region.

FIG. 9 is a depiction of a representative tail region.

FIG. 10 is a depiction of a representative tail region.

FIG. 11 is a depiction of a representative tail region.

FIG. 12 is a depiction of a representative tail region.

FIG. 13 is a depiction of the general structure of agents of the currentinvention in which the core region is connected to a tail region.

FIG. 14 is a depiction of an agent of the current invention, designatedAgent A.

FIG. 15 is a depiction of an agent of the current invention, designatedAgent B.

FIG. 16 is a depiction of an agent of the current invention, designatedAgent C.

FIG. 17 is a depiction of an agent of the current invention, designatedAgent D.

FIG. 18 is a depiction of an agent of the current invention.

FIG. 19 is a depiction of an agent of the current invention.

FIG. 20 is a depiction of an agent of the current invention.

FIG. 21 is a schematic diagram of the synthetic process for the agentdepicted in FIG. 4.

FIG. 22 is a schematic diagram of the synthetic process for Agent C,depicted in FIG. 16.

FIG. 23A and FIG. 23B are depictions of the molecules added during thesynthetic processes shown in FIGS. 21 and 22. FIG. 23C is a schematicdiagram of a synthetic process for the molecule shown in FIG. 23B.

FIG. 24 is a schematic diagram for the synthetic process for Agent A,depicted in FIG. 14, in which R is hydrogen.

DETAILED DESCRIPTION OF THE INVENTION

The current invention pertains to a series of agents, depicted in FIGS.2-6 and 14-20, which represent a new class of agents for stabilizationof poliovirus and related viruses, and also for antiviral activityagainst poliovirus and related viruses. The agents were computationallybased on several X-ray structures of poliovirus. A computer program wasused to create functional (chemical) group maps of the Sabin 3poliovirus binding site. Selected functional group minima were connectedby energy minimization and dynamics to obtain agent molecules.

The agents have a "core" region which fits deepest into the drug bindingpocket of the virus, and can also have a more variable "tail" region.The general structure of the core region is depicted in FIG. 1, and thegeneral structure of the tail region is depicted in FIG. 7. The uniquefeatures of the core region are two fused double aromatic rings (U andW), which consist of either a five-membered aromatic ring fused to asix-membered aromatic ring, or two six-membered aromatic rings fused toone another; and a single six membered ring (V) which optionally canhave a hydroxyl group substituent on any of the available ring atoms.The two fused double aromatic rings are connected by single linkercarbons, such that the core region has the structure:

    U--CH.sub.2 --V--CH.sub.2 --W.

One or more atoms in the two fused double aromatic rings can beheteroatoms (nitrogen or oxygen), substituted for carbon atoms. Examplesof core regions of agents of the current invention are shown in FIGS.2-6. The core region occupies most of the drug binding site.

The features of the tail region include a polar group (X) having anoxygen atom, a positively charged group (Y), and a bulky fused doublearomatic ring structure (Z) at the end, which consists of either afive-membered aromatic ring fused to a six-membered aromatic ring, ortwo six-membered aromatic rings fused to one another. This fused doublearomatic ring may have one or more heteroatoms (nitrogen or oxygen)substituted for carbon atoms. Both the polar group (X) and thepositively charged group (Y) interact with the protein. In oneembodiment of the current invention, the positively charged group is anammonium group. These functional groups are arranged such that the tailregion has the structure:

    X--Y--Z.

The tail region extends out of the drug binding site into the receptorbinding site.

If a tail region is present, it is connected to the core region by acovalent linkage (such as a methylene or chain of methylenes) such thatthe fused double aromatic ring W is connected to polar group X. Thestructure of an agent having both a core region and a tail region is:

    U--CH.sub.2 --V--CH.sub.2 --W--X--Y--Z.

FIG. 13 depicts the general structure of an agent having a core regionand a tail region. Examples of such agents are shown in FIGS. 14-20.

Any of the functional groups (U, V, W, X, Y, and/or Z) can additionallyhave small groups attached. Such groups include alkyl (such as methyl[--CH₃ ]) groups; halides (such as fluorine or chlorine); hydroxyl(--OH) groups; or amino (--NH₂) groups.

The fused double aromatic rings at either end of the core region inthese agents are not present in any currently existing antipicornavirusdrugs; furthermore, they are more rigid and compact than the groupswhich are present in the other drugs. A striking difference betweenthese agents and existing drugs is the placement of a bulky group at thecenter of the binding pocket, specifically the phenol group between thetwo fused double aromatic rings of the core region. In addition, thepresence of a relatively long tail region is in itself unique.

The agents of the current invention were designed using computationalmethods. Computational agent design can be thought of as a three stepprocess. First, the localization of specific functional (chemical)groups in the binding site is calculated, such that the groups havepotential energy minima. Second, the resulting functional group minimaare clustered and connected to design new ligands or agents for thebinding pocket. Third, the binding constants of new ligand are estimatedto predict how tightly the ligand will bind.

In an example of computational agent design (described in further detailin Example 2 below), the Multiple Copy Simultaneous Search (MCSS)program (Miranker, A. and Karplus, M., Proteins, 11:29 (1991)) has beenused to "map" the agent binding site of poliovirus. This work was thefirst application of MCSS for designing non-peptide ligands. Theresulting minima were clustered and connected to form two new agents forpoliovirus, shown in FIGS. 14 (Agent A) and 15 (Agent B).

The agents of the current invention can be used to stabilize capsids ofpicornaviruses and other related viruses by contacting the virus withthe agent. The agents can thus be used to stabilize unstable forms ofvirus for experimental studies. The agents can also be used to increasethe stability, particularly the thermal stability, of existingpoliovirus vaccines or vaccines for related viruses. In addition, theagents can be used to prevent viral changes necessary for cell entry.The agents can also be used to terminate replication of live vaccinevirus after sufficient time has elapsed to induce an immune response inan individual, minimizing the risk of vaccinees shedding reverted viruswhich are neurovirulent. The agents can additionally be used forprophylactic treatment of non-vaccinated family members of vaccinees.The agents can further be used for prophylaxis and therapeutic treatmentof infection with enterovirus, Coxsackievirus, echoviruses, and otherpicornaviruses with accessible binding sites.

The ability of the agent to bind virus can be assessed by determiningthe MIC (minimum inhibitory concentration) value. The MIC value is foundfor these agents using standard methods (Andries, K. et al., J. Virol.64:1117-1123 (1990)). Briefly, serial dilutions of the agent are addedto solutions containing approximately 100 tissue culture infectiousdoses of viruses, and the resulting mixtures are added to subconfluentlayers of HeLa cells in a microtiter plate. The MIC is taken as thelowest concentration of agent that protects 50% of cells from cytopathiceffect (death).

Standard organic chemistry methods can be used to produce the agents ofthe current invention. For example, FIG. 21 depicts the molecules in thesteps of the synthetic process for the agent depicted in FIG. 4, whichis also the core region of Agents C and D. FIG. 22 depicts the moleculesin the steps of the synthetic process for Agent C. The molecules addedduring the synthetic processes are depicted in FIGS. 23A and 23B. Thesemolecules can be derived from known compounds; for example, the moleculeshown in FIG. 23A can be derived from 5-hydroxyisophthalic acid (Aldrich#31,127-7). The molecule shown in FIG. 23B can be derived fromDL-2-pyrrolidone-5-carboxylic acid (Aldrich #29,291-5) or fromDL-glutamic acid; alternatively, it can be synthesized by the pathwaysshown in FIG. 23C. Standard reaction conditions are used (see, forexample, March, Advanced Organic Chemistry (3d edition), Wiley & Sons,New York, N.Y. 1985); Organic Syntheses, Wiley & Sons, New York, N.Y.(annual volumes); Buehler and Pearson, Survey of Organic Synthesis,Wiley & Sons, New York, N.Y. (1970, 1977)). For alkylation of indoles inthe 3-position by alcohols which can generate carbonium ions, seeHellman, H. et al., Ann. Chem. 604:214 (1957); under basic conditions,see Pratt, E. F. et al., J. Am. Chem. Soc. 79:5248 (1957), and Johnson,H. E., U.S. Pat. No. 3,197,479; see also Cardillo, B., et al.,Tetrahedron 23:3771 (1967). For synthesis of anaminoalkylphenylenediamene, see Piotrovskii, L. B. et al., Khim-Fram.Zh. 9(10): 305 (1975). For synthesis of indoles substituted in thesix-membered ring with β-aminoethyl groups, see Troxler, F. et al.,Helv. Chim. Acta 51:1616 (1968). For synthesis of indoles substituted inthe six-membered ring with formyl groups, see Backenberg, O. G. et al.,J. Chem. Soc. 3961 (1962). Potential starting materials include:5-hydroxy-isophthalic acid (Aldrich 31,127-8); 5-formylsalicylaldehyde(Aldrich 27,534-4) (4-hydroxyisophthalaldehyde); 5-formylsalicylic acid(Aldrich F1,760-1); DL-2-pyrrolidone-5-carboxylic acid (Aldrich29,921-5); DL-glutamic acid (Aldrich G279-6); L-glutamic acid 5-methylester (Aldrich 85,829-9); and 5-cyanoindole (Aldrich C9,200-6)(5-indolecarbonitrile).

The invention is further illustrated by the following Examples, whichare not to be considered limiting in any way.

EXAMPLE 1

Viral Structure and Virus/Agent Complex Structure

A. The structure of poliovirus and related picornaviruses

Poliovirus is a nonenveloped, icosahedral virus with a single-stranded,plus-sense RNA genome of approximately 7500 nucleotides enclosed in aroughly spherical protein capsid of approximately 310 Å in diameter anda maximum thickness of 50 Å. The viral capsid consists of 60 copies eachof the viral proteins VP1, VP2, VP3, and VP4 for a total molecularweight of 8.5×10⁶ daltons. The virus coat-proteins VP1, VP2, and VP3 areeach eight-stranded wedge-shaped antiparellel β-barrels with twoflanking helices, while VP4 is a short peptide. The immature particle iscomposed of the three viral proteins VP0, VP1 and VP3; duringmaturation, VP0 is cleaved to yield VP4 and VP2. In the mature particle,the viral proteins form a protomer unit with VP1, VP2, and VP3 packedtogether in pseudo T=3 positions and VP4 on the back side of theprotomer inside the capsid. Protomers pack around the local five-foldaxis to form pentamers and then twelve pentamers form the viral capsid.It is possible experimentally to isolate pentamers, and also emptycapsids composed of VP0, VP1 and VP3.

The narrow end of the VP1 β-barrels packs around the five-fold axes,while the narrow ends of VP2 and VP3 alternate around the three-foldaxes of the particle. Several prominent loops connecting the β-barrelsof VP1, VP2 and VP3 are on the surface of the viral capsid and includeresidues identified as parts of antigenic sites (Emini, E. A., et al.,J. Virol. 43:997 (1982); Crainic, R., et al., Infect. Immun. 41:1217(1983); Minor, P. D., et al., Nature 301:674 (1983); Minor, P. D., etal., J. Gen. Virol. 65:1159 (1985); Ferguson, M., et al., Virology143:505 (1985); and Diamond, D. C., et al., Science 229:1090 (1985)).Examination of the pentamer unit reveals a depression on the capsidsurface or a "canyon" around the bottom of the β-barrels of the fivesymmetry-related copies of VP1, and it is proposed that cellularreceptors bind to the virus in this canyon (Olson, N. H., et al., Proc.Natl. Acad. Sci. USA 90:507 (1993); and Hogle, J. M., Cur. Biol. 3:278(1993)). At the base of the canyon there is an opening to a binding sitewithin the β-barrel of VP1 that can accommodate various substituents. Inall existing poliovirus structures there is electron-density in thisbinding pocket, and in the P1/Mahoney and P3/Sabin structures thedensity has been convincingly modeled as the lipid sphingosine. This isthe same site that is occupied by the existing drugs, including the R(Janssen) and WIN (Sterling-Winthrop) compounds, in both poliovirus andin rhinovirus (Rossman, M. G., et al., Nature 317:145 (1985); and Kim,K. H., et al., J. Mol. Biol. 230:206 (1993)). The native structures ofrhinovirus 1A and 16 have a short-chain fatty acid modeled into thepocket, while the higher resolution rhinovirus 14 structure shows onlyfour waters bound in the pocket (Smith, T. J., et al., Science 233:1286(1986)). When the Sterling-Winthrop antiviral drug WIN 51711 binds torhinovirus 14 (Smith, T. J., et al., Science 233:1286 (1986)), part ofthe "GH loop" (between β-strands G and H) in VP1 moves over 4 Å andpartially "closes" the entrance to the pocket. This "trap-door"(residues 1232 to 1237 in poliovirus) is in essentially this same closedposition in all poliovirus and rhinovirus structures that have eithernatural substituents or drugs in the binding pocket. In poliovirus, thecarboxyl end of the GH loop of VP1 makes contact with the GH loop of VP3which is near the protomer interface. Although early studies withrhinovirus 14 implicated the large conformational changes observed upondrug binding as being important in the antiviral activity of theSterling-Winthrop "WIN" drugs, the lack of structural changes upon drugbinding in rhinovirus 1A and rhinovirus 16, suggests an alternativemechanism in which occupation of the site interferes with conformationalchange associated with receptor binding (in rhinovirus 14 and 16).

Antiviral drugs prevent a variety of conformational transitions of thevirus, including those necessary for productive cell entry (McSharry, J.J., et al., Viroloqy 97:307 (1979)) such as cell attachment and capsiduncoating, and therefore it seems likely that these drugs are exploitinga site that is normally used to regulate the stability of enteroviruses.Presumably the existing, partially effective drugs (such as theSterling-Winthrop compound WIN 51711 and the Janssen compound R78206),displace the natural substituents because they have a higher bindingconstant and this tighter binding may prevent required rearrangements ofthe virus either by making the capsid too stable or by causingconformational changes upon binding that later interfere with cellattachment.

B. Existing virus/drug complex crystal structures

Several high resolution X-ray structures of drug complexes withpoliovirus and rhinovirus exist (Zhang, A., et al., Virology 3:453(1992)). They include the structures of P3/Sabin poliovirus complexedwith each of the Janssen compounds R78206, R80633, and R77975; themutant P3/Sabin poliovirus F1124L/F1134L (these binding site residuesare Leu in the Mahoney strain) complexed with the Janssen compoundR78206; and rhinovirus 14 complexed with a number of Sterling-Winthropcompounds and Janssen compounds (Zhang, A., et al., Virology 3:453(1992)). The Janssen compounds differ only in the number of linker --CH₂groups connecting the methyl-pyridozinyl piperidine group to thebenzoate group. In all of the Janssen structures these drugs areoriented with the pyridazine group deepest in the pocket and thebenzoate group nearest to the entrance. Similarly, the Sterling-Winthropcompound WIN 51711 is oriented so that the double ring, oxazoline groupis deepest into the β-barrel and the isoxazoline group is near theentrance. In other Sterling-Winthrop WIN compounds, the occupancy of thedrug binding site is reversed, such that the isoxazoline group isdeepest into the β-barrel and the double ring is near the entrance. Theoccupancy of the Janssen compounds in the poliovirus binding site isgreatest for R78206 (about 90%) and diminished for R80633 and again somefor R77975.

EXAMPLE 2

Computational Agent Design

Most of the previously existing drugs for poliovirus and the relatedrhinovirus consist of two single, non-fused rings connected by a longchain to two additional single, non-fused, rings. In the preferredembodiment, new agents effective in early intervention againstpoliovirus and rhinovirus should have MIC values at least as low asthose of the existing drugs. To determine which region of the virusstructure defines the binding site and needs to be included in designcalculations, the five drug complex structures described above, as wellas the native P3/Sabin and P1/Mahoney poliovirus structures, werecompared. Lists were compiled of all capsid residues within 4.5, 6, 8,10, and 12.5 Å, respectively, of the substituent in the protomer 1binding site in any of the six poliovirus structures described above.Several protomer 2 (which is related to protomer 1 by a 72° rotationabout the five-fold axis) residues are near the protomer 1 binding site,and therefore must be considered in all agent design calculations.

A. Computational methodology used for agent design

As a first step in the design process, functional group maps of theP3/Sabin poliovirus binding site were made using the computer programMCSS (Miranker, A. and Karplus, M., Proteins, 11:29 (1991)). To preparethe protein coordinate set for the calculations, polar hydrogens wereadded to the P3/Sabin protomer using the hbuild command in CHARMM(Brooks, B. R., et al., J. Comp. Chem. 4:187 (1983)) and standardPARAM19 parameters and topology, and then a symmetry operation wasapplied to create the adjacent pentamer. The coordinate set was editedto include only those residues with atoms within 12.5 Å of the ligand inany of the six polio structures described above, plus five residues oneither side of each of those residue ranges. It is necessary to excisepart of the protein, because although the protein is fixed during anMCSS calculation, the number of protein atoms does significantlyincrease the CPU time required. The binding site is defined as anapproximately 20×20×30 Å³ box that would enclose the sphingosine inprotomer 1 of the native P3/Sabin poliovirus structure.

In a typical MCSS run, N copies of a given functional group wererandomly distributed in the specified binding site, where N is usuallybetween 1000 and 5000. Functional groups are typically simple smallmolecules. A large number of functional groups are available in thecurrent implementation of MCSS, and additional functional groups caneasily be included. The N copies of the group were then simultaneously,and independently, energy minimized in the field of the fixed protein,using a modified version of the program CHARMM. By the time-dependentHartree approximation (Elber, R., et al., J. Am. Chem. Soc. 112:9161(1990)), each copy felt the full force field of the protein but thecopies did not interact with each other. More specifically, the N copiesof the group were simultaneously subjected to 500 steps of steepestdecent minimization followed by 500 steps of Powell minimization, andthen nine cycles of 1000 steps of Powell minimization each, for a totalof 10,000 minimization steps. After every 1000 steps of minimization,the functional group minima were gathered to remove duplicate minima.Minima were also deleted from the system after each cycle except for thefirst, if their interaction with the protein energy was too high asdetermined by a series of user specified energy cutoffs. After the finalcycle, the remaining minima were sorted by interaction energy and theircoordinates and interaction energy were written to a file. Since theprotein competes with solvent for binding functional groups, only minimawhose free energy of binding to the protein was less than their freeenergy of solvation were considered. In the Born approximation, one halfof the enthalpy of solvation is equal to the free energy of solvationfor charged ions, (Roux, B., et al., J. Phys. Chem. 94:4683 (1990)) andtherefore for charged and polar functional groups only minima with aninteraction energy less than one half their solvation enthalpy wereexamined. Numerous test calculations were performed mapping the groupN-methyl acetamide into the P3/Sabin binding site, to determine the setof protein residues, number of copies of a group, anddistance-to-protein cutoff for the initial distribution of the copies,and gather cutoffs to use for the final series of MCSS calculations withvarious functional groups.

B. Mapping various functional groups into the P3/Sabin poliovirusbinding site

Several polar, charged, aromatic, and aliphatic functional groups havebeen mapped into the P3/Sabin poliovirus binding site, includingN-methyl acetamide, methanol, water, acetic acid, methyl-ammonium,unhydrated Mg²⁺, MG²⁺. . . H₂ O (treated as one functional group), thetryptophan sidechain, the histidine sidechain, phenol, benzene, thephenylalanine sidechain, cyclohexane, propane, and isobutane. For thisprotein the cpu time required to minimize 1000 group copies ranged fromabout 11 hours for a small group like methyl-ammonium to about 60 hoursfor a large group like the tryptophan sidechain on a singe SGI R3000processor. These calculations predicted that a pattern of doublesix-membered (or five- and six-membered fused) aromatic ring connectedto a single six-membered aromatic ring connected to a double ring againshould preferentially bind in the P3/Sabin poliovirus binding site.Also, a side-pocket at the protomer-protomer interface was identified asa possible alternative drug binding site. The results showed that thisside-pocket, which branches off the center of the main binding pocket,could accommodate a ligand with net positive charge.

C. Clustering and connecting functional group minima

After mapping the various functional groups into the binding site, theminima were clustered by inspection and a few of the best (lowestenergy) minima were selected from each cluster. A set of these minimawas then connected by placing linker --CH₂ groups between the minimawhere necessary to create a chemically sensible molecule. First, thelinker atoms were minimized and annealed in the fixed protein with aminima also fixed in their MCSS positions. Then, the entire newlydesigned agent molecule was minimized in the fixed protein. Finally, theagent molecule and the protein were minimized together, and later theinteraction energy of the minimized agent with the fixed protein wascalculated. Through this process, two new agent molecules were designed;these are shown in FIGS. 14 and 15. To facilitate synthesis, Agent A wasmodified and Agents C and D were modeled through the process describedabove. Energy minimizations of the agent/virus complexes suggest thatthe agents will bind at least as strongly as the best of the currentlyknown compounds. All four agents have significantly lower interactionenergy with the protein than the Janssen compound R78206; these resultsare summarized in Table 1, below.

                  TABLE 1                                                         ______________________________________                                        Interaction Energy of Agents.sup.1                                                       Agent       Agent      Agent                                                  with links  minimized in                                                                             minimized                                   Total of   optimized in                                                                              fixed      with                                        mcss       fixed protein                                                                             protein (vdw                                                                             protein (vdw                                minima.sup.2                                                                             (vdw and elec.sup.2)                                                                      and elec.sup.2)                                                                          and elec.sup.2)                             ______________________________________                                        Agent          305.4       -57.8    -121.7                                    A     (-166.6) (41.0)      (-117.5) (-181.8)                                  Agent          1657.5      -22.2    -104.0                                    B     (-167.5) (1400.5)    (-106.6) (-180.1)                                  Agent --       458.1       -58.6    -136.6                                    C              (96.9)      (-124.0) (-197.4)                                  Agent --       669.8       -68.6    -136.5                                    D              (268.9)     (-130.5) (-191.1)                                  R78206                                                                              --       --          -4.0     -40.2                                                                (-35.9)  (-66.0)                                   ______________________________________                                         .sup.1 All energies are in kcal/mol and include the internal energy terms     of the drug unless otherwise stated.                                          .sup.2 Includes van der Waals (vdw) and electrostatic (elec) energy terms     only.                                                                    

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

What is claimed is:
 1. A compound, wherein the compound is representedby a structure selected from the group consisting of: ##STR1## wherein Ris --H or --OH.